PROJECT SUMMARY Congenital heart defects affect a devastating 0.8-1.2% of live human births, largely due to anatomical malformations of the heart. However despite vast advancements in our knowledge of genetic control over congenital defects, because of the complexity of developmental morphogenesis the mechanical cause of these malformations largely remains unknown. During human embryonic development, a variety of signaling cues trigger multicellular morphogenic movements involved in primitive heart formation. However, attempts to address human developmental processes have been limited due to notably divergent embryonic processes, even between mammalian embryos, and the inability of current human in vitro models of tissue development to control the emergence of heterogeneous cell populations required for specialized morphogenic processes. In particular, changes in intercellular adhesion molecules are essential to all stages of developmental morphogenesis, including mesoderm emergence, neural crest establishment, heart field migration, and heart septa formation, all of which are required for cardiac morphogenesis and specification. Essential for cardiac development are epithelial-to-mesenchymal transitions (EMT) that drive subpopulation motility. Consequently, EMT dysregulation can result in human embryonic heart defects. A vital component of EMT is the reduction of adhesion molecule E-cadherin (Ecad), which has been connected to several key developmental signaling programs (canonical Wnt, TGF?, and Hippo) through its recruitment of the protein ?-catenin to cell-cell contacts. However, how changes in Ecad adhesions alter ?-catenin's ability to transcriptionally regulate these three pathways has yet to be interrogated directly. As a result, fundamental questions about how Ecad coordinates human morphogenic events and lineage fate are still unanswered. The proposed research will attempt to design a human in vitro model system in human induced pluripotent stem cells that will model morphogenic processes driven by the heterogeneous regulation of Ecad adhesions. This allows for the manipulation of patterning events that mimic aspects of EMT and subsequently interrogate unanswered questions about early human developmental morphogenic processes. In summary, interrogation of Ecad regulation of morphogenic lineage fate decisions will be approached by completing three specific aims: (1) Determine whether heterogeneous loss of E-cadherin is sufficient to drive phenotypes characteristic of EMT; (2) Determine how heterogeneous loss of E-cadherin associated with EMT affects mesodermal lineage specification; and (3) Interrogate how differential E- cadherin alters ?-catenin transcriptional activity. Ultimately, the proposed research will assess how adhesion changes coordinate and direct EMT processes and lineage specification, which has the potential to address mechanisms behind species-specific embryogenesis and morphogenesis, and more importantly, to provide insights into the development of congenital heart defects.